Title: Zona incerta GABA neurons modulate energy homeostasis Abstract. Obesity, which often leads to secondary health complications including heart disease, diabetes, stroke, cancer, and early death, has become a major health concern in the US. Here we test the general hypothesis that neurons in the rostromedial zona incerta (ZI), and particularly inhibitory GABA neurons, play an unexpectedly profound orexigenic role in increasing food intake and body weight. Most of the work done on the neuronal regulation of energy homeostasis has previously focused on neurons in other brain regions. The first Aim examines the structural substrates for interaction between ZI GABA axons and their postsynaptic targets. Using confocal scanning laser microscopy and dual immunolabel electron microscopy coupled with cre recombinase-dependent AAV and rabies virus tracers, we test the hypothesis that ZI axons project to a number of sites, including the paraventricular thalamus (PVT) and hypothalamic ventromedial nucleus (VMH) where direct synaptic connections are made with excitatory neurons. We use multiple transgenic mouse lines expressing Cre recombinase under control of various neuron-selective promoters including mice that express Cre in GABA neurons driven by a vGAT promoter. These will be coupled with intracerebral microinjections of AAV viral vectors containing floxed GFP or tdTomato reporter genes to study ZI GABA neuron efferent and afferent axon projections. We will also employ injection of a Cre recombinase-dependent Brainbow-type pseudorabies virus into the ZI; after retrograde axonal transport this PRV expresses a red reporter in wild-type cells, but in Cre-expressing GABA cells, reporter expression changes to yellow or cyan, helping define the cell of interest. Aim 2 tests the hypothesis that ZI GABA cells respond to long distance signals of energy homeostasis including ghrelin and leptin, and also to axonally released neuropeptide modulators of food intake. Whole cell recording allows us to test different mechanisms of action on ZI GABA cells produced by neuromodulator signals from other neurons involved in energy homeostasis. C-fos expression will be examined after food deprivation to test the hypothesis that ZI GABA neurons are more active during reduced food availability. Optogenetics is used in brain slices to test the hypothesis that release of transmitter from ZI GABA axons will exert similar inhibitory effects on PVT and VMH neurons; neighboring ZI dopamine cells are also tested. In Aim 3, we examine the role that rostromedial ZI GABA neurons play in ongoing energy homeostasis by cell silencing (using Gi-DREADDs and caspase) to test the hypothesis that body weight and food intake is reduced. Optogenetic activation with ChR2 variant ChIEF will test the hypothesis that stimulation of ZI GABA axons in different terminal zones will each enhance food intake and body weight. We also test the hypothesis that ZI GABA neuron activation provides a positive emotional valence. Together, these experiments examine converging structural, electrophysiological, and behavioral analyses, focusing on the role of the GABA ZI neurons in energy homeostasis. With the growing levels of obesity in this country approaching 30% of the adult population, and the associated health complications, identifying and understanding the brain cells that control and sense energy homeostasis will help to identify novel approaches to reducing the trend toward obesity.